[Subarachnoid Hemorrhage and Isolated Brainstem Death]. / Aneurysmatische Subarachnoidalblutung und Entwicklung eines "isolierten Hirnstammtods".

BACKGROUND: Worldwide there are differences in the procedure of determining brain death. An irreversible loss of all brain functions, including cerebrum, cerebellum and brainstem is mandatory for the diagnosis of brain death in Germany. On the basis of a case report some important aspects of the new recommendations of the German guidelines are discussed. CASE REPORT: We present the case of a 41-year old patient who was admitted to our clinic due to acute subarachnoid hemorrhage (SAH). Angiography revealed an aneurysm of the posterior inferior cerebellar artery. The patient was comatose without any brainstem reflexes and showed apnoea. However, on day 3, EEG showed alpha activity as a sign of residual cortical function. We diagnosed an isolated brainstem death. The next day EEG was isoelectric and brain death was confirmed. DISCUSSION: The diagnosis of isolated brainstem death does not allow a confirmation of death in Germany. Our case presents a primary infratentorial brain damage mandating additional confirmatory tests.

Craniocervical manual lymphatic drainage and its impact on intracranial pressure - a pilot study.

BACKGROUND AND PURPOSE: Theoretical considerations and the results of animal studies indicate that manual lymphatic drainage (MLD) might have an impact on intracranial pressure (ICP). There is a lack of clinically qualitative investigations on patients with severe cerebral diseases. METHODS: Between April 2013 and January 2015 a prospective observational study was performed on patients who were undergoing intracranial pressure measurement and treatment with MLD. ICP, cerebral perfusion pressure, mean arterial pressure (MAP), heart rate and oxygen saturation were recorded continuously 15 min before the procedure, during MLD (22 min) and for 15 min after the procedure. For analysis the data treatment units were divided into two groups: patients with a mean baseline ICP <15 mmHg (group 1) and patients with a mean ICP ≥15 mmHg before MLD (group 2). RESULTS: A total of 133 treatment units (61 patients) were analysed (group 1 n = 99; group 2 n = 34). The mean baseline ICP was 10.4 mmHg overall, and 8.3 mmHg and 18.6 mmHg respectively in group 1 and group 2; ICP significantly decreased during therapy with MLD and this persisted during the follow-up period in group 2. MAP did not show any significant differences between the different periods. CONCLUSIONS: Our data showed a significant reduction of ICP during therapy with craniocervical MLD in patients with severe cerebral diseases.

Dysphagia due to triple A syndrome: successful treatment of achalasia by balloon dilatation.

UNLABELLED: Triple A syndrome is a rare autosomal recessive inherited disorder which is characterized by alacrima, adrenal insufficiency, and achalasia. We report on a 14-year old girl with dysphagia, regurgitation, and vomiting since 5 years. At the age of five years an Addison crisis was diagnosed and cortisone substitution was initiated. In addition, the patient had episodes of conjunctivitis. Severe esophagitis and candida infection were diagnosed by esophago-gastro-duodenoscopy and treated with omeprazole and fluconazole. The esophageal barium swallow was typical for achalasia. Medical treatment of achalasia with oral nifedipine resulted only in a partial and temporal improvement. But after seven balloon dilatations dysphagia and nocturnal coughing improved clearly and a remarkable gain of weight could be seen. Direct sequencing showed a homozygous nonsense mutation in exon 11 of the AAAS gene leading to truncation at position 342 of the 546 amino acid protein. CONCLUSION: Triple A syndrome has to be considered in patients with dysphagia. In our patient, the absence of tears since birth followed by adrenal insufficiency were early signs of the triple A syndrome. Balloon dilatation of the esophago-gastric junction is an effective treatment, which can avoid surgical interventions.

Sleep-wake disturbances in sporadic Creutzfeldt-Jakob disease.

BACKGROUND: The prevalence and characteristics of sleep-wake disturbances in sporadic Creutzfeldt-Jakob disease (sCJD) are poorly understood. METHODS: Seven consecutive patients with definite sCJD underwent a systematic assessment of sleep-wake disturbances, including clinical history, video-polysomnography, and actigraphy. Extent and distribution of neurodegeneration was estimated by brain autopsy in six patients. Western blot analyses enabling classification and quantification of the protease-resistant isoform of the prion protein, PrPSc, in thalamus and occipital cortex was available in four patients. RESULTS: Sleep-wake symptoms were observed in all patients, and were prominent in four of them. All patients had severe sleep EEG abnormalities with loss of sleep spindles, very low sleep efficiency, and virtual absence of REM sleep. The correlation between different methods to assess sleep-wake functions (history, polysomnography, actigraphy, videography) was generally poor. Brain autopsy revealed prominent changes in cortical areas, but only mild changes in the thalamus. No mutation of the PRNP gene was found. CONCLUSIONS: This study demonstrates in sporadic Creutzfeldt-Jakob disease, first, the existence of sleep-wake disturbances similar to those reported in fatal familial insomnia in the absence of prominent and isolated thalamic neuronal loss, and second, the need of a multimodal approach for the unambiguous assessment of sleep-wake functions in these patients.

Role-playing for more realistic technical skills training.

Clinical skills are an important and necessary part of clinical competence. Simulation plays an important role in many fields of medical education. Although role-playing is common in communication training, there are no reports about the use of student role-plays in the training of technical clinical skills. This article describes an educational intervention with analysis of pre- and post-intervention self-selected student survey evaluations. After one term of skills training, a thorough evaluation showed that the skills-lab training did not seem very realistic nor was it very demanding for trainees. To create a more realistic training situation and to enhance students' involvement, case studies and role-plays with defined roles for students (i.e. intern, senior consultant) were introduced into half of the sessions. Results of the evaluation in the second term showed that sessions with role-playing were rated significantly higher than sessions without role-playing.

[Cranial irradiation induces premature activation of the gonadotropin-releasing-hormone]. / Schädelbestrahlung verursacht vorzeitige Aktivierung des Gonadotropin-Releasinghormon (GnRH)-Pulsgenerators bei Ratten - Ein neuesTiermodell für strahleninduzierte Störungen der Pubertät.

BACKGROUND: CNS-irradiation in prepubertal children with leukemia or brain tumors can lead to precocious or in high doses to delayed puberty. The underlying mechanisms of these disorders are unknown. METHODS: A new animal model of experimentally induced pubertal disorders by cranial irradiation has been developed. In infantile or juvenile (12 - 23 days old) female rats precocious or delayed puberty have been induced by selective cranial Co60-irradiation (4 - 18 Gy). At age of 32 - 38 days or 3 months relevant hormone parameters have been studied basal and after stimulated conditions. RESULTS: Low radiation doses (5 or 6 Gy) led to accelerated onset of puberty as well as elevated LH- and estradiol levels. High radiation doses (9 - 18 Gy) caused retardation of sexual development, lower gonadotropin levels and growth retardation associated with growth hormone deficiency. After cranial irradiation with 5 Gy the release rates of the inhibitory neurotransmitter gamma-aminobutyric-acid (GABA) from hypothalamic explants were significantly lower (p < 0,05). The gonadotropin-releasing-hormone (GnRH) expression in the hypothalamic preoptic area of irradiated animals (5 Gy) was significantly higher than in controls (p < 0,05). CONCLUSION: The GnRH-pulse generator is very radiosensitive as low dose irradiation causes precocious puberty, whereas high dose irradiation is associated with delayed sexual maturation. Radiation induced precocious puberty might be caused by damage to inhibitory GABAergic neurons leading to desinhibition and premature activation of GnRH neurons. Our animal model of cranial irradiation seems to be suitable to study neurotransmitter disorders, molecular mechanisms and potential preventive intervention of radiation induced pubertal changes.

[The iodine supply of newborns. Comparison of iodine absorption and iodine excretion of mother and child]. / Die Jodversorgung von Neugeborenen. Vergleich der Jodaufnahme und Jodausscheidung von Mutter und Kind.

BACKGROUND: Since 1989 the use of iodized salt has been allowed in Germany, additional supplementation with iodide tablets has been recommended during pregnancy and lactation. This study was undertaken to clarify whether the iodine intake of neonates and young infants improved since then. PATIENTS AND METHODS: In the first part of the study the urinary iodine excretion of 52 newborns and their mothers in 1998 was compared to data of similar studies 1983 in the area of Göttingen and 1982 in the areas of Heidelberg and Rothenburg, Germany. All these are geographically low-iodine areas. In the second part the iodine supply of infants in 1998-1999 under feeding with mother's milk or formulas in 1998 and 1999 was obtained by measuring iodide concentrations in urine and milk using a high pressure liquid chromatography (HPLC) method. RESULTS: 45% of pregnant women were without iodide supplementation in 1998. In 1998 the median urinary iodide concentration during the first week of life was 4.3 micrograms/dl, which was more than twice that found in 1983 (1.75 micrograms/dl). Infants feeding by mother's milk without maternal iodine supplementation or by semi-elementary diet had the lowest urinary iodine excretion, whereas significantly higher values were measured when feeding formulas for term or preterm infants. CONCLUSIONS: The iodine intake of newborns has markedly improved during 15 years. The WHO criterias for adequate iodine supply (TSH < 5 microU/ml and urinary iodine >/ = 10 micrograms/dl) were only partly fulfilled in Göttingen indicating that a mild iodine deficiency still exists with the risk of iodine deficiency disorders.

Autoregulation of the gonadotropin-releasing hormone (GnRH) system during puberty: effects of antagonistic versus agonistic GnRH analogs in a female rat model.

To address whether gonadotropin-releasing hormone (GnRH) regulates its own expression and the expression of its receptor in the hypothalamus and ovary, we treated five groups of prepubertal/peripubertal female rats from postnatal days 25-36 with either the GnRH agonist triptorelin (TRIP) or the GnRH antagonist cetrorelix (CET), each 10 or 100 microgram/day, or a placebo. We compared their effects regarding pubertal development, serum gonadotropins and the expression of GnRH and GnRH-receptor in the hypothalamus, pituitary, ovary and uterus. Onset of puberty was determined by vaginal opening, and expression levels of GnRH and GnRH-receptor were determined using either quantitative real-time PCR or competitive RT-PCR. Onset of puberty was retarded by both analogs but CET (100 microgram/day) inhibited while TRIP (10 and 100 microgram/day) stimulated serum gonadotropins (P<0.05). The expression of GnRH in the preoptic area did not show significant differences among the treatment groups but ovarian GnRH mRNA levels were significantly stimulated by CET (100 microgram/day). GnRH mRNA could not be detected in the uterus by either real-time PCR or competetive RT-PCR. The GnRH-receptor expression in the hypothalamus (preoptic area and mediobasal hypothalamus) did not vary among any of the groups, whereas in the pituitary GnRH-receptor mRNA levels were stimulated by TRIP (10 microgram/day) but inhibited by CET (100 microgram/day). In contrast, in the ovary GnRH-receptor mRNA levels were inhibited by both TRIP (100 microgram/day) and CET (100 microgram/day). Interestingly, the GnRH-receptor was even expressed in the uterus where it was strongly stimulated by both CET and TRIP in a dose-related manner. This shows that in addition to their different pituitary effects, the GnRH analogs cetrorelix and triptorelin exert different actions at the hypothalamic, ovarian and uterine level. This study also demonstrates an organ-specific regulation of GnRH and GnRH-receptor gene expression which is likely part of a local autoregulatory system. We conclude that the ovarian and uterine effects of GnRH analogs must be considered in addition to their known pituitary effects when deciding which GnRH analog is most suitable for treating precocious puberty.

Reduction of gamma-aminobutyric acid-ergic neurotransmission as a putative mechanism of radiation induced activation of the gonadotropin releasing-hormone-pulse generator leading to precocious puberty in female rats.

Brain irradiation in prepubertal children with malignomas can cause precocious puberty. A selective cranial cobalt (Co(60))-irradiation technique has been developed in rats. In two experiments early juvenile (13-15 days old) female rats received a single dose of 5 Gy or sham irradiation. At pubertal age (post-natal days 33-34) irradiated rats had higher serum estradiol and luteinizing hormone levels. In experiment 1 irradiated rats had higher gonadotropin releasing-hormone (GnRH) mRNA levels in the preoptic area compared to controls (P<0.05). In experiment 2 the release rates of gamma-aminobutyric acid (GABA) in vitro from preoptic mediobasal hypothalamic areas of irradiated rats were significantly reduced after stimulation with the GABA(A) receptor agonist muscimol (maximum values 4607+/-804 vs. 7399+/-1048 pM in controls, mean+/-SEM, P<0.05). Radiation induced central precocious puberty might be caused by damage to inhibitory GABAergic neurons leading to premature activation of the GnRH-pulse generator.

GnRH antagonist cetrorelix prevents sexual maturation of peripubertal male rats.

UNLABELLED: Gonadotropin releasing-hormone (GnRH) analogues contain amino acid substitutions of the native decapeptide. Depending on the substitutions, the analogues have GnRH agonistic or antagonistic properties. GnRH agonists are the established treatment in cases of central precocious puberty caused by premature activation of the hypothalamic GnRH pulse generator. Much less data exist on the use of GnRH antagonists to influence the onset of puberty. Using the GnRH antagonist cetrorelix we conducted a 5 day treatment of peripubertal male rats (cetrorelix group n=12, 100 microg/d intraperitoneally injected; placebo n=10, NaCl 0.9% intraperitoneally injected) from postnatal day 32 to 36 and decapitated on postnatal day 37 to investigate the effects on pubertal development, serum gonadotropin and testosterone levels as well as the GnRH release from explanted hypothalami. A control group of 5 male rats was added for hypothalamus superfusion experiments on day 25. We observed no progress of testicular development in the cetrorelix group. Cetrorelix injected rats had lower testicular weights (531+/-13 versus controls 819+/-25 mg, mean+/-SEM, p<0.0001). 12 h after the last injection testosterone levels were in the castrate range (serum testosterone, median controls: 1.7 ng/ml, median cetrorelix <0.30 ng/ml, p<0.001), and they showed lower serum LH and FSH compared to the same age placebo group. After decapitation the preoptic mediobasal hypothalamic area (POA/MBH) was dissected from 5 randomly selected rats from each treatment group and the release rates of GnRH were determined in superfusion experiments: The hypothalamic GnRH secretion was comparable in the CET and the same age placebo rats but significantly higher than in the 25 day old control group. CONCLUSION: The GnRH antagonist cetrorelix inhibits the pituitary-gonadal axis in peripubertal male rats and may be effective in treating central precocious puberty in males.

Effects of medial thalamotomy and pallido-thalamic tractotomy on sleep and waking EEG in pain and Parkinsonian patients.

OBJECTIVES: Investigation of sleep and sleep EEG before and after stereotactic neurosurgery. METHODS: All-night polysomnographic recordings were obtained in 3 neurogenic pain patients and 3 parkinsonian patients. One subject of each group was recorded in addition 3 months after surgery. Stereotactic operations were performed in the medial thalamus and on the pallido-thalamic tract to relieve neurogenic pain and parkinsonian symptoms, respectively. RESULTS: Sleep efficiency was little affected by the surgical intervention in neurogenic pain patients and a dramatic reduction in REM sleep occurred, which had recovered in the subject recorded after 3 months. After the surgery parkinsonian patients showed an increase in total sleep time and in sleep efficiency, and a decrease in REM sleep latency. Sleep efficiency remained elevated in the 3 months follow-up. Medial thalamotomy abolished spindle frequency activity (SFA) in the power and coherence spectra in non-REM sleep stage 2 systematically. Pallido-thalamic tractotomy attenuated SFA only to varying degrees. After 3 months SFA had reemerged. The alpha peak of the waking EEG was shifted to lower frequencies after surgery in 5 of 6 patients and had reverted to the original frequency 3 months later. CONCLUSIONS: Medial thalamotomy or pallido-thalamic tractotomy had acute and reversible effects on the EEG and long-term deleterious side effects of stereotactic surgery on sleep and sleep EEG are improbable. The results provide further evidence for the involvement of the human thalamus in the generation of sleep spindles.

Genomic structure and DNA binding properties of the human zinc finger transcriptional repressor AP-2rep (KLF12).

We recently cloned a novel murine transcriptional repressor, the Krüppel-like zinc finger protein AP-2rep (HGMW-approved symbol KLF12), that binds to a regulatory element in the AP-2alpha gene promoter. In the present study, we characterize the human AP-2rep homolog and describe expression patterns in human urogenital and lymphoma cell lines. The predicted human protein of 402 amino acids exhibits 95.8% identity and 98.5% similarity to the murine AP-2rep peptide. The genomic locus of human AP-2rep consists of seven exons and was assigned to chromosome 13q22 by fluorescence in situ hybridization to metaphase chromosomes. Human AP-2rep repressed both reporter expression from a transiently transfected AP-2alpha promoter and the endogenous AP-2alpha gene and inversely was negatively regulated by AP-2alpha. The consensus motif CAGTGGG was identified by an in vitro binding site selection assay. In summary, our data further point to an important role of AP-2rep as a transcriptional silencer and reveal reciprocal regulation of AP-2alpha and AP-2rep.

Transcriptional regulation of the AP-2alpha promoter by BTEB-1 and AP-2rep, a novel wt-1/egr-related zinc finger repressor.

AP-2 transcription factors have been suggested to exert key regulatory functions in vertebrate embryonic development, in tumorigenicity of various cancer cell types, and in controlling cell cycle and apoptotic effector genes. In this study, we investigated transcriptional regulation of the AP-2alpha gene promoter mediated by an autoregulatory element (referred to as A32) with a core consensus AP-2 binding site at position -336 relative to the mRNA initiation site. AP-2 and multiple different nuclear proteins in HeLa and Neuro2A cell extracts form specific bandshifts with the A32 element. By screening a mouse brain cDNA expression library, we isolated two different cDNAs encoding the transcription factor BTEB-1 and a novel zinc finger protein, AP-2rep. AP-2rep reveals a modular structure with homology to transcription factors of the wt-1/egr-1-family. AP-2rep, BTEB-1, and AP-2 interact in a mutually exclusive manner with overlapping binding sites in the A32 element. Transfection studies revealed that BTEB-1 is a strong activator of AP-2alpha promoter activity, whereas cotransfected AP-2alpha resulted in moderate autoactivation of promoter activity. In contrast, AP-2rep confers strong transcriptional repression to the AP-2alpha gene, and we observed an excellent correlation between induction of AP-2rep mRNA expression and downregulation of AP-2alpha mRNA during development of the kidney. In summary, we have identified multiple transcription factors and cloned from an expression library a novel zinc finger silencing factor, AP-2rep, mediating positive and negative regulation of AP-2alpha expression through a set of overlapping cis-regulatory promoter elements.

Ontogeny of the GNRH-, glutaminase- and glutamate decarboxylase-gene expression in the hypothalamus of female rats.

Amino acid neurotransmitters like gamma-aminobutyric acid (GABA) and glutamate (GLU) are involved in the regulation of hypothalamic gonadotropin releasing hormone (GnRH) release. We investigated, whether there are changes of gene expression in the rat hypothalamus for GnRH, GnRH receptor, as well as glutaminase and glutamate decarboxylase, two enzymes regulating neurotransmitter concentrations of GLU and GABA in the brain during the ontogeny. After reverse transcription-polymerase chain reaction (RT-PCR) we used an ELISA method to quantify PCR products. In 15-day old animals high plasma luteinizing hormone (LH) levels with pronounced variations were found. In 25-day old animals LH values were low, whereas in 35-day old rats LH levels increased significantly indicating the reactivation of the GnRH-pulse generator at the beginning of puberty. In parallel to these changes, the mRNA levels of the GnRH receptor in the mediobasal hypothalamus were high at day 15, significantly lower at day 25 and again high at day 35 after birth (ELISA O.D. GnRH-R day 15: 0.46+/-0.07, day 25: 0.16+/-0.04, day 35: 0.36+/-0.04; p<0.01), but no changes of GnRH receptor gene expression were found in the preoptic area. The mRNA of GnRH in the preoptic area as well as mRNA levels of glutaminase and glutamate decarboxylase in the mediobasal hypothalamus and the preoptic area did not change during ontogeny. We conclude that hypothalamic GnRH receptors are involved in the characteristic changes of LH secretion patterns during sexual maturation. Major changes of GnRH receptor gene expression occurred in the mediobasal hypothalamus and correlated well with plasma LH levels, whereas hypothalamic mRNA levels of GnRH, glutaminase and glutamate decarboxylase did not change within the different age groups. Thus the activity of the GABA- and glutamatergic system during ontogeny may be regulated at the receptor or postreceptor level.

Short stature and failure of pubertal development in thalassaemia major: evidence for hypothalamic neurosecretory dysfunction of growth hormone secretion and defective pituitary gonadotropin secretion.

UNLABELLED: In patients with beta-thalassaemia major, frequent blood transfusions combined with desferrioxamine chelation therapy lead to an improved rate of survival. Endocrine disorders related to secondary haemosiderosis such as short stature, delayed puberty and hypogonadism are major problems in both adolescent and adult patients. A total of 32 patients with beta-thalassaemia major undergoing treatment at the Children's Hospital, University of Göttingen were examined. Fourteen of these were short in stature. Growth hormone (GH) secretion was investigated in 13 patients exhibiting either a short stature or reduced growth rate. The stimulated GH secretion of 10 patients in this subgroup lay within the normal range. Studies of their spontaneous GH secretion during the night revealed that these patients had a markedly reduced mean GH and reduced amplitudes in their GH peaks. Low insulin-like growth factor (IGF)-I levels were seen in the growth-retarded thalassaemic patients. Eight were subjected to an IGF generation test and showed a strong increase in both IGF-I and insulin-like growth factor binding protein (IGFBP)-3 levels indicating intact IGF-I generation by the liver. Hypogonadotropic hypogonadism was found to be present in both the male and female patients with impaired sexual development. After priming with LH-releasing hormone (GnRH) per pump in 2 female and 5 male patients, no change in either their serum oestradiol or testosterone levels or in LH/FSH response to GnRH was observed suggesting that they were suffering from a severe pituitary gonadotropin insufficiency. Three male patients at the age of puberty but exhibiting short stature. low GH, low IGF-I and hypogonadism received low dose long-acting testosterone. After 3 12 months of therapy there was a marked growth spurt, higher nocturnal GH levels and an increase in both IGF-I and IGFBP-3. CONCLUSION: Reduced GH secretion and low IGF-I in thalassaemic patients are related to a neurosecretory dysfunction due to iron overload rather than to liver damage. Hypogonadotropic hypogonadism is caused by the selective loss of pituitary gonadotropin function. In patients with both GH deficiency and hypogonadism, low dose sexual steroid treatment should be considered either as an alternative or an additional treatment before starting GH therapy.

Involvement of gamma amino butyric acid (GABA) in the postnatal function of the GnRH pulse generator as determined on the basis of GnRH and GnRH-receptor gene expression in the hypothalamus and the pituitary.

In many species the GnRH pulse generator functions early postnatally to become arrested during infancy. In rats highly variable LH levels in 15-day-old animals are suggestive that LH is being released by the pituitary in pulses whereas between day 20 after birth and puberty LH levels are low indicating that the GnRH pulse generator is arrested. In the present study we show on the basis of consecutively withdrawn blood samples in 15-day-old animals that LH pulses are indeed present at that age. The proper function of GnRH receptors in the pituitary is crucially dependent on pulsatile GnRH release from the hypothalamus. In addition, GnRH receptors have been demonstrated in the medial preoptic area and in the mediobasal hypothalamus of adult rats. In 15-day-old animals the functional GnRH pulse generator results in upregulated GnRH receptor gene expression as demonstrated by quantitative RT-PCR. It is not known what neural mechanisms are involved in turning the GnRH pulse generator off during infancy and a GABAergic brake has been discussed. Indeed, when 30-day-old animals were injected with the GABA-A receptor blocking drug bicuculline, this resulted in increased serum LH levels indicating that a tonic GABAergic inhibition is indeed operative at this age.

Effects of dihydrotestosterone alone and combined with estrogen on bone mineral density, bone growth, and formation rates in ovariectomized rats.

Androgens are associated with the greater skeletal mass and size in men compared with women and have been used as anabolic agents promoting skeletal growth and mineral accretion in both sexes, but specific effects on growth and bone formation in the female skeleton are not well understood. The effects of 5 alpha-dihydrotestosterone (DHT) alone, and in combination with 17 beta-estradiol on bone and bone growth were studied in female ovariectomized (OVX) rats with established osteopenia. Eight weeks after OVX, rats were given 0.1 mg 17 beta-estradiol and/or 2.5 mg or 10 mg DHT administered by controlled-release pellets for 2 months. Body weights decreased with estrogen treatment but increased with DHT. Bone mineral density increased with the highest dose of DHT relative to OVX controls and the estrogen treated group. Dry and ashed bone weights and ash/dry weight ratios increased in the estrogen and DHT treated animals compared to the baseline OVX controls. Total bone calcium was greater with DHT and estrogen combined with DHT. The percent of calcium in the ash increased in all DHT treated groups. When normalized to final body weight, the total femur calcium content was significantly increased in the estrogen and estrogen with DHT groups, but not in the DHT groups compared with the baseline OVX and OVX control groups. The periosteal bone formation rates were increased with the high dose DHT alone and combined with estrogen. OVX rats had increased endochondral bone elongation rates relative to controls but this was decreased with estrogen treatment. DHT combined with estrogen increased endochondral growth rates relative to the estrogen treated group. Trabecular bone volume was decreased in all OVX groups relative to the base line group, but there were no significant effects observed with any treatments. Cancellous bone formation rates were suppressed with estrogen treatment but were partially reversed when combined with DHT. DHT treatments also increased most cancellous bone formation indices over OVX controls. While estrogen is known to preserve skeletal mass by reducing bone turnover, DHT increased skeletal mass by promoting bone growth and formation with concomitant increases in total body mass. DHT had greater effects on cortical bone and partially mitigated the suppressive effects of estrogen on bone growth and formation in the female skeleton.

Systemic antitumor effects of electrochemotherapy combined with histoincompatible cells secreting interleukin-2.

Electrochemotherapy is an antitumor treatment that combines a cytotoxic drug with the local administration of electric pulses delivered at the tumor site. We previously found that in mice the cure rate of subcutaneous transplanted tumors treated by electrochemotherapy is increased by repeated systemic interleukin-2 (IL-2) injections. Moreover, histoincompatible cells engineered to secrete IL-2 allow the rejection of syngeneic tumor cells when both cells are inoculated together. In this study of preestablished tumors in mice we show that after electrochemotherapy, delayed peritumoral injections of histoincompatible IL-2-producing cells result in the cure of almost all the tumors. Moreover, this combined local treatment leads to cures of untreated, contralaterally transplanted tumors. This systemic antitumor immunity also resulted in complete protection of the cured mice against further inocula of the tumor cells. These results, which were obtained using allogeneic as well as xenogeneic IL-2-secreting cells, suggest that electrochemotherapy combined with such cellular immunotherapy might be a useful approach for the treatment of metastasizing cancers.